ProjectSummary: As the most common inherited blood disorder in the United State, there are 70,000-100,000 Americans with sickle cell anemia. Sickle cell disease (SCD) is caused by a mutation in the ?-globin gene, which leads to significant deformation red blood cell (RBC) membrane and promotes RBC adhesion to other cells to induce vaso-occlusive crises (VOC). Chronic sickle cell anemia is accompanied with progressive systemic multi- systemic organ dysfunction and cost over $475 million annually in hospital admission. Our laboratory has reported that sickle cell-induced hypoxic, oxidant, and inflammatory stress is perpetuated by aged neutrophils whichpositivelycorrelateswithVOCinhumanizedSCDmice.Ourrecentworkhasdemonstratedthatdeletion ofthemicrobiotainSCDmicebyantibioticsrestrictedagedneutrophilexpansionwhichconsequentlydecreased VOCseverity,andreducedorgandamageaswellastheironoverload.Inaddition,hydroxyurea,theonlyFDA- approved drug for SCD that promotes the anti-sickling fetal hemoglobin expression, also possesses anti- inflammatory,antiradical,andmetal-chelatingactivitiesbothinmammaliancellsaswellasinbacteria. Inthisapplication,weproposea3-yearexperimentalplanthatwilladvanceourunderstandinginthefunctionof microbiota in SCD disease progression and will test whether manipulation of the microbiota will provide a potentialnovelSCDtreatment.InSpecificAim1,wewillidentifydisease-modifyingmicrobiotaspeciesthatmay contributetoneutrophilagingandSCDorgandamage.16Ssequencingdatahasrevealedmicrobiotadifferences betweenantibiotictreatmentanduntreatedSCDmice,andthefunctionofselectedmicrobiotawillbeverifiedin germ-freeSCDmice.InSpecificAim2,wewillexamineifhydroxyureareducesVOCandorgandamagethrough microbiotamanipulationsinSCDmiceinwhichfecalsamplesfromhydroxyurea-treatedandcontrolSCDmice will be transplant to germ-free SCD to evaluate whether hydroxyurea-induced changes in the microbiota contributetoitstherapeuticactivity.InSpecificAim3,wewillstudyifironrestriction(byDFOorlow-irondiet)or probiotics-inducedchangesinironmetabolismcanamelioratechronicorgandamageinSCDmice.Iron-related changesinmicrobiotawillbeverifiedbygerm-freeSCDmice.Theseproposedstudies,focusedonstrategiesof microbiota manipulation in SCD, will allow us to identify the key microbial species that contribute to SCD pathophysiology, and provide potential novel cost-effective approaches for management of SCD?s life-long complications.